KR20230031804A - Composition for decomposing pesticides - Google Patents

Abstract

The present invention relates to a composition for decomposing pesticides, which, by using at least one selected from a group consisting of microorganisms of the genus Achromobacter, a culture solution of the microorganisms, the concentrated solution of the culture solution and the dried product of the culture solution as active ingredients, can decompose pesticides in an eco-friendly way to be effective in protecting ecosystem.

Description

Composition for decomposing pesticides

The present invention relates to a composition for decomposing pesticides using microorganisms.

Chemical pesticides have contributed greatly to the improvement of agricultural productivity, but due to indiscriminate use, they cause various environmental problems such as soil and water pollution and residues on crops. In order to overcome these problems, most pesticides in Korea are managed through safety evaluation and pesticide use guidelines. Nevertheless, the problem of transfer of pesticides used in previous crops to subsequent crops frequently occurs, resulting in enormous damage to farmhouses. Some of the pesticides sprayed in agricultural fields are attached to crops or permeate into the air, and most of them settle in the soil. Pesticide components in the soil are naturally degraded, but in the case of non-degradable pesticides, the decomposition process is very complicated and takes a long time. Therefore, when a large amount of pesticides accumulate in the soil, not only soil contamination, but also permeation into groundwater and affecting rivers and lakes are increasing concerns about the destruction of soil and aquatic ecosystems. Therefore, it can be said that it is urgent to establish an efficient system that can quickly decompose pesticides remaining in the soil of harvested fields.

Accordingly, studies on biodegradation of pesticides using useful microorganisms for agriculture have been steadily conducted as part of reducing the burden on the agricultural environment and ecosystem and at the same time supplying safe food. Bacteria, such as the genus Bacillus and Pseudomonas , and fungi, such as the genus Aspergillus and the genus Penicillium , are known as the main microorganisms used to decompose biological pesticides. These microorganisms are Not only harmless to crops and humans, but also without causing direct contamination of the soil, decomposing pesticides remaining in the soil to protect the ecosystem, while preventing the transfer of residual pesticides to soil and crops.

However, in the case of these various pesticide biodegradable microorganisms, it is not easy to actually apply them to the field, and especially carbamate-based pesticides have not been studied in depth on their biodegradability, so development of biodegradable microorganisms for carbamate-based pesticides This is what is needed.

An object of the present invention is to provide a composition capable of decomposing pesticides in an environmentally friendly manner.

In order to achieve the above object, one aspect of the present invention is for decomposing pesticides comprising at least one selected from the group consisting of a microorganism of the genus Achromobacter , a culture solution of the microorganism, a concentrate of the culture solution, and a dried product of the culture solution. composition is provided.

In addition, in order to achieve the above object, another aspect of the present invention provides a method for removing residual pesticides comprising the step of treating the composition for decomposing pesticides in an environment in which pesticides are expected to remain.

Microorganisms of the genus Acromobacter used as an active ingredient in the composition for decomposing pesticides of the present invention are not only harmless to crops and humans, but also show activity to decompose pesticides remaining in soil, especially carbamate-based pesticides, and are therefore environmentally friendly. It has the effect of protecting the ecosystem.

1 is a phylogenetic diagram analyzing the phylogenetic position of the GHC2-5 strain of the present invention.
2 is an experimental result confirming the carbofuran degradation activity of the GHC2-5 strain of the present invention.
3 is an experimental result confirming the decomposition activity of the GHC2-5 strain of the present invention against other carbamate-based pesticides.
4 is an experimental result confirming the decomposition activity of the GHC2-5 strain of the present invention against other mixed carbamate-based pesticides.
5 is an experimental result confirming the carbofuran decomposition activity of other microorganisms of the genus Acromobacter, in addition to the GHC2-5 strain of the present invention.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a composition for pesticide decomposition.

The composition for decomposing pesticides of the present invention includes at least one selected from the group consisting of a microorganism of the genus Achromobacter , a culture solution of the microorganism, a concentrate of the culture solution, and a dried product of the culture solution.

The microorganisms of the genus Achromobacter are Achromobacter arsenitoxydans , Achromobacter cholinophagum, Achromobacter cycloclastes , Achromobacter denitrificans ( Achromobacter denitrificans ), Achromobacter fischeri, Achromobacter insuavis, Achromobacter hartlebii , Achromobacter immobilis, Achromobacter immobilis , Achromobacter insolitor ( Achromobacter insolitus ), Achromobacter lactolyticus ( Achromobacter lactolyticus ), Achromobacter agrifaciens ( Achromobacter aegrifaciens ), Achromobacter lyticus ( Achromobacter lyticus ), Achromobacter methanolophila ( Achromobacter methanolophila ), Ark Achromobacter pestifer , Achromobacter piechaudii, Achromobacter ruhlandii , Achromobacter marplatensis , Achromobacter spanius ( Achromobacter spanius ), Achromobacter viscosus , Achromobacter xerosis , Achromobacter xylosoxidans , and the like, Achromobacter insu abis, Achromobacter agri Perciens, Achromobacter piecoaudii, Achromobacter xyloxoxydans, Achromobacter spanius, It may be Acromobacter pestifer, Acromobacter marplatensis, and the like, and in particular, it may be Acromobacter pestifer, preferably Achromobacter pestifer GHC2-5 deposited under accession number KACC 92347P.

The culture solution is obtained by culturing the microorganism of the genus Achromobacter, and may be a culture solution itself containing cells of the microorganism, or a concentrate or dried product thereof, but is not limited thereto, and the composition is treated within the environment of the ecosystem. Anything processed into any form may be included as long as it is included so that the microorganism of the genus Achromobacter can survive and grow or have an activity.

The concentrate may be obtained by increasing the concentration of solids in the culture medium, and by increasing the cell concentration of microorganisms of the genus Acromobacter. The concentrate may be concentrated by vacuum concentration, plate type concentration, thin film concentration, etc., but is not limited thereto. The content of the culture medium included in the composition of the present invention can be appropriately adjusted according to the concentration of the concentrate.

The dried product includes, but is not limited to, those dried through methods such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high frequency drying, and infrared drying. For example, when using a freeze-dried product obtained by freeze-drying a culture solution containing cells of microorganisms belonging to the genus Achromobacter, there are advantages in formulating, packaging, and storing a composition containing the same, and it can be stored for a long period of time while being arc-free. It has the advantage of not damaging the biological activity of microorganisms of the genus Lomobacter.

The pesticide may include a carbamate-based pesticide, and the carbamate-based pesticide may include, for example, aldicarb, aldoxycarb, allyxycarb, and bendiocarb. , benfuracarb, benthiocarb, carbaryl, carbofuran, carbosulfan, chlorpropham, diethofencarb, ethienocarb (ethienocarb), ethiofencarb, fenobucarb, fenoxycarb, formetanate, furathiocarb, isoprocarb, methiocarb ( methiocarb, methomyl, methomyl-oxime, metolcarb, oxamyl, pirimicarb, propamocarb, propham ), propoxur, terbucarb, thiodicarb, XMC (3,5-Xylyl methylcarbamate), xylylcarb, etc., especially carbaryl, carbofuran, and ethiophencarb , fenovucarb, methiocarb, propoxer, and the like.

The pesticide decomposition composition may further include a suitable nutrient source so that the microorganisms of the genus Acromobacter can survive until directly used for pesticide decomposition. Appropriate nutrient sources that may be included in the composition may be used without particular limitation as long as they are known to those skilled in the art as necessary for the survival of microorganisms, and specifically may be glucose, maltose, galactose, sugar, lactose or starch as a carbon source, but Not limited.

The composition for decomposing pesticides may be formulated in various forms. For example, it may be prepared in the form of directly sprayable solutions, powders and suspensions or in highly concentrated aqueous, oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, dusting materials or granules, but is not limited thereto. In this case, appropriate solvents and/or carriers may be included as needed, and inactive additives or surface-active substances such as emulsifiers or dispersants may also be included.

In addition, another aspect of the present invention provides a method for removing pesticide residues.

The method for removing residual pesticides includes treating the composition for decomposing pesticides in an environment in which pesticides are expected to remain.

The environment may be any environment as long as it is expected to be contaminated with pesticides, and in particular, it may be an ecosystem such as soil or water, but is not limited thereto.

Leachate, runoff, aquifers, groundwater, surface water, well water, soil, agricultural or industrial samples, and/or pesticides in various environments such as industrial ponds, wastewater treatment facilities, water sources with mosquito ponds, etc. A variety of carbamate pesticides can be detoxified, decontaminated, altered, eliminated or reduced.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[1-1] preparation of pesticides

Carbaryl, carbofuran, ethiofencarb, fenobucarb, methiocarb and propoxur from Sigma-Aldrich (St, Louis, MO, USA) were purchased, and they were mixed with acetone (Merck, USA) to a final concentration of 10,000 ppm, respectively, and stored in a refrigerator.

[1-2] Sample collection and enrichment culture

In a cabbage field in Goheung-gun, Jeollanam-do, which has been continuously treated with chemical pesticides for many years, 50 g of soil samples were collected at a depth of 5 to 10 cm after skimming 1 to 2 cm of topsoil. The soil sample collected as described above was used in the experiment after removing large-sized stones and foreign substances with a 2 mm sieve (Daihan Standard Test Sieve; Daihan Co., Korea).

In a sterilized 250 ml glass flask, MSM (minimal salt media) pH 7.5 (potassium phosphate dibasic 7.5 g/L, potassium phosphate monobasic 2 g/L, magnesium chloride 0.5 g/L and sodium chloride 0.5 g/L 49.5 mL, trace metal solution (ammonium molybdate 0.02 g/L, cobalt chloride hexahydrate 0.003 g) /L, boric acid 0.05 g/L, zinc chloride 0.03 g/L, copper(II) acetate monohydrate 0.01 g/L and iron(II) chloride chloride) 0.02g/L) into 500μL, and 5g of soil sample prepared as above and 1% (v/v) of 10,000ppm carbofuran solution prepared in Example [1-1] were added and shaken. It was cultured for 2 weeks at 120 rpm at 25° C. in a shaking incubator (Vision Co., Korea) (primary enrichment culture). Then, 5 ml of the culture medium of the primary enrichment culture was added, and subculture was performed twice under the same conditions and methods as those of the primary enrichment culture.

[1-3] Microbial isolation and identification

After serial dilution of the enriched culture in Example [1-2] by 1/10 in sterile distilled water (3M, USA), 100 μL of a 1:1000 diluted sample was added to the R2A plate medium (Lab M , UK) and incubated for 12-15 hours in an incubator at 30 ° C. Then, the microbial colony visible to the naked eye as dominant bacteria was streaked on the R2A plate medium to isolate the pure GHC2-5 strain. The GHC2-5 strain isolated as described above was inoculated into 5 mL of R2A liquid medium, cultured for 15 hours, mixed with 80% glycerol solution at a ratio of 3:1, and stored at -70°C.

The GHC2-5 strain isolated as described above was sent to Cheonlab Co., Ltd. for 16S rRNA sequence analysis and genome sequence analysis, and the obtained sequence was analyzed through EzBioCloud (https://www.ezbiocloud.net/). Meanwhile, the phylogenetic position of the GHC2-5 strain was determined using the MEGA X program. As a result, as shown in FIG. 1A, the GHC2-5 strain was classified into the genus Achromobacter and confirmed to exhibit the highest 16S rRNA sequence homology with Achromobacter pestifer. It became. When considering the homology of these nucleotide sequences and the phylogeny, it is determined that the GHC2-5 strain belongs to Achromobacter pestifer, but is a genetically distinct strain. Accordingly, the GHC2-5 strain was deposited with the Korean Agricultural Culture Collection (KACC) on May 6, 2021, and was given accession number KACC 92347P.

Confirmation of pesticide decomposition activity

[2-1] Confirmation of carbofuran degradation activity

The GHC2-5 strain frozen in Example [1-3] was smeared on R2A plate medium and cultured for 12 to 15 hours, then inoculated into 10 mL of R2A liquid medium and 12-150 rpm at 30 ° C. It was pre-incubated for 15 hours.

500 μL of the 10,000 ppm carbofuran solution prepared in Example [1-1] was added to 50 mL of R2A broth, and the pre-culture solution pre-cultured as described above was inoculated at a concentration of 2% (v/v). and cultured with shaking for 4 to 5 days at 150 rpm at 30 ° C.

In the process of shaking culture as described above, 10 mL of the culture medium was aliquoted every 24 hours, and they were put together with 10 mL of acetonitrile (Duksan, Korea) in a 50 mL conical tube and observed for 30 seconds. After vortexing, additional QuEChERS powder mix (magnesium sulfate 4g, sodium chloride 1g, trisodium citrate dihydrate 4g, disodium hydrogen citrate sesquihydrate) 0.5 g) was added and vortexed for 30 seconds, followed by centrifugation at 4° C. at 10,000 rpm for 10 minutes. Then, 5mL of the supernatant was obtained with a 10mL syringe, filtered through a syringe filter (0.2m), and the filtrate obtained as above was stored at -70°C.

The solvent of the filtrate was evaporated using a centrifugal vacuum concentrator (HyperVac, GYROZEN, Korea) connected to a cooling trap device (HyperCool, GYROZEN, Korea), and the solid component remaining in the tube was suspended in 20 μL of acetone to prepare a 100-fold concentrate. Then, it was diluted 1/1000 with acetonitrile and analyzed by performing TLC (thin layer chromatography). A TLC silica plate (Aluminium HPTLC Silica gel 60 F ₂₅₄ plates, Merck, USA) was cut to a size of 5 cm × 5 cm or 5 cm × 10 cm according to the purpose of the experiment and used for the experiment, and the TLC developing solvent was used for simple development of the filtrate. In , a solvent in which ethyl acetate and hexane were mixed at a ratio of 50:50 was used. The optical density (OD600) was measured with a spectrophotometer (Libra S50, Biochrom, UK) set to a wavelength of 600 nm, and the LC-MS/MS used in this experiment was an AB Sciex 5500 triple-quadrupole mass spectrometer (AB Sciex, Toronto Canada) equipped with Agilent 1260 series (Agilent Technologies, Wilminton, DE), Osaka Soda CAPCELL CORE C18 (150 mm × 2.1 mm, 2.7 μm, Shiseido, Japan) was used as a column, and A: 0.1% mobile phase was used. Aqueous solution containing formic acid and 0.5 mM ammonium formate, B: 99% methanol solution containing 0.1% formic acid and 0.5 mM ammonium formate, flowing at a flow rate of 0.3 mL/min and the temperature of the column oven was set to 40°C. LC-MS/MS was analyzed in ESI (electron spray ionization) positive mode to measure the residual amount of pesticide. In addition, in order to verify the test method under the obtained LC-MS / MS instrument analysis conditions, each pesticide standard was diluted with acetonitrile to prepare samples with concentrations of 0.02, 0.05, 0.1, and 0.2 ppm, respectively, and 2 μL was subjected to LC-MS / MS was injected into the concentration to confirm the linearity, and a calibration curve was drawn up.

As a result, as shown in FIG. 2, the concentration of carbofuran in the culture medium for culturing the GHC2-5 strain gradually decreased over time, and on the 5th day of culture, carbofuran almost did not exist in the culture medium. found to be decomposed.

[2-2] Confirmation of degradation activity for other carbamate pesticides

In order to confirm whether the GHC2-5 strain exhibits degrading activity against carbamate-based pesticides other than carbofuran as in Example [2-1], the carbaryl prepared in Example [1-1] , Ethiophencarb, Fenobucarb, Methiocarb, and Propoxer solutions were used, and culture and analysis were performed in the same manner as in Example [2-1].

As a result, as shown in FIG. 3, it was confirmed that the GHC2-5 strain of the present invention exhibits the same decomposition activity not only for carbofuran but also for other types of carbamate-based pesticides.

In addition, the same method as in Example [2-1] using a mixed solution in which 20 ppm of each of the five carbamate-based pesticides (carbaryl, etiophencarb, fenovucarb, methiocarb, and propoxer) was mixed Culture and analysis were carried out.

As a result, as shown in Figure 4, it was confirmed that the GHC2-5 strain of the present invention exhibits the same decomposition activity not only for carbofuran, but also for a composition in which five other carbamate-based pesticides are mixed.

Confirmation of the activity of other microorganisms of the genus Achromobacter

In addition to the GHC2-5 strain identified in Example 2, other strains belonging to the genus Achromobacter were also confirmed to have an activity to decompose carbamate-based pesticides.

To this end, Achromobacter piechaudii , KACC 12987), Achromobacter xylosoxidans , KACC 14136, Spanius ( Achromobacter spanius , KACC 16377) and Achromobacter marplatensis (KACC 16929) was distributed, and inoculated in R2A broth containing a 100 ppm carbofuran solution in the same manner as in Example [2-1] The assay was performed while culturing.

As a result, as shown in Table 1 and FIG. 5, in addition to the GHC2-5 strain of the present invention, the concentration of carbofuran in the culture medium for culturing other strains belonging to the genus Achromobacter gradually decreases over time, On the 5th day of culture, it was confirmed that the amount of carbofuran in the culture medium was significantly reduced.

Time CNT (Carbofuran + R2A) A . piechaudii A . xylosoxidans A . spanius A . marplatensis 0 100% 100% 100% 100% 100% 24 100.1% 99.19% 99.85% 91.89% 95.69% 48 98.87% 76.15% 89.36% 81.07% 51.34% 72 88.39% 55.26% 89.61% 61.18% 72.74% 96 88.54% 20.37% 51.86% 33.49% 42.94% 120 84.11% 12.66% 38.59% 20.26% 28.78%

From the above results, it can be seen that strains belonging to the genus Achromobacter, including the GHC2-5 strain of the present invention, have an activity to decompose carbamate-based pesticides in common.

In the above, representative experimental examples of the present invention have been illustratively described, but the scope of the present invention is not limited to the specific experimental examples as described above, and those skilled in the art are within the scope described in the claims of the present invention. will be able to change accordingly.

Claims (7)

Acromobacter ( Achromobacter ) A microorganism, a culture solution of the microorganism, a composition for degrading pesticides comprising at least one selected from the group consisting of a concentrated solution of the culture solution and a dried product of the culture solution. The method of claim 1,
Microorganisms of the genus Achromobacter are Achromobacter insuavis , Achromobacter aegrifaciens , Achromobacter piechaudii , Achromobacter xylosoxi Dans ( Achromobacter xylosoxidans ), Achromobacter spanius ( Achromobacter spanius ), Achromobacter pestifer ( Achromobacter pestifer ) or Achromobacter marplatensis ( Achromobacter marplatensis ) A composition for degrading pesticides. The method of claim 2,
Wherein the Achromobacter pestifer is Achromobacter pestifer GHC2-5 deposited under accession number KACC 92347P. The method of claim 1,
The pesticide is a pesticide decomposition composition comprising a carbamate-based pesticide. The method of claim 4,
The carbamate pesticides are aldicarb, aldoxycarb, allyxycarb, bendiocarb, benfuracarb, benthiocarb, carbaryl ), carbofuran, carbosulfan, chlorpropham, diethofencarb, ethienocarb, ethiofencarb, fenobucarb, fenoxycarb, formetanate, furathiocarb, isoprocarb, methiocarb, methomyl, methomyl-oxime , metolcarb, oxamyl, pirimicarb, propamocarb, propham, propoxur, terbucarb, thiodicarb ( thiodicarb), XMC (3,5-Xylyl methylcarbamate) and xylylcarb (xylylcarb) at least one selected from the group consisting of pesticide decomposition composition. The method of claim 5,
The carbamate pesticide is selected from the group consisting of carbaryl, carbofuran, ethiofencarb, fenobucarb, methiocarb and propoxur At least one composition for decomposing pesticides. A method for removing residual pesticides comprising: processing the composition for decomposing pesticides according to any one of claims 1 to 6 in an environment in which pesticides are expected to remain.

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